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Abstract

Background— Pitavastatin (NK-104) is a novel inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme for cholesterol biosynthesis. In clinical trials, pitavastatin has been shown to significantly decrease serum LDL cholesterol and triglyceride levels and increase HDL cholesterol. Scavenger receptor-mediated accumulation of oxidized LDL (OxLDL)-derived cholesteryl ester is considered to be a critical step in the development of atherosclerotic foam cell formation. We studied the effect of pitavastatin on CD36 (a class B scavenger receptor) expression by murine macrophages.

Received October 31, 2002; de novo received August 5, 2003; revision received October 1, 2003; accepted October 6, 2003.

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors or statins are potent inhibitors of cholesterol biosynthesis. Clinical trials have provided clear evidence that cholesterol-lowering therapy with this class of compounds decreases the incidence of coronary heart disease.1 However, the overall clinical benefits observed with statin therapy are greater than what might be expected from changes in lipid profile alone. This suggests that the beneficial effects of statins may extend beyond their effects on serum cholesterol levels.1–3 Experimental and clinical evidence indicates that some of the cholesterol-independent effects of statins involve (1) endothelial normalization of nitric oxide production,4,5 (2) antiinflammatory effects and inhibition of monocyte/endothelial cell adhesion,6,7 (3) inhibition of scavenger receptor expression,8,9 (4) strengthening of the fibrous cap,10,11 (5) inhibition of platelet thrombus formation/reduction of thrombotic response,12 and (6) inhibition of smooth muscle cell proliferation.13,14

Scavenger receptors are thought to play a significant role in atherosclerotic foam cell development because of their ability to bind and internalize modified lipids, such as oxidized LDL (OxLDL).15,16 Two major classes of human scavenger receptors, designated types A and B, have been identified. CD36, the defining member of type B scavenger receptors, binds OxLDL but not LDL.17 CD36 cDNA-transfected cells bind and internalize OxLDL, and binding of OxLDL to human macrophages was 50% blocked by antibodies to CD36.18 Both native and modified lipids upregulate expression of the class A19 and class B20 scavenger receptors. Inhibition of CD36 expression has been demonstrated to reduce the development of atherosclerosis in atherosclerosis-prone apolipoprotein E-null mice.21

Recent studies have implicated peroxisome proliferator-activated receptor-γ (PPARγ) in the regulation of CD36 by oxidized lipoprotein.22,23 PPARγ is a member of the nuclear hormone receptor superfamily. PPARγ heterodimerizes with the retinoid X receptor (RXR) and functions as a transcriptional regulator of genes that modulate lipid metabolism and adipocyte gene expression.24 PPARγ is activated by such diverse agents as long-chain fatty acids, arachidonic and linoleic acid metabolites,25 and the thiazolidinedione class of antidiabetic drugs.26 The phosphorylation status of PPARγ has been shown to affect its transcription activity. PPARγ is a positive regulator for its target genes, whereas its phosphorylated form (PPARγ-Pi) is a negative regulator.27

Pitavastatin, also known as NK-104 (CAS 147526-32-7; monocalcium bis [(3R, 5S, 6E)-7-(2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl)-3,5-dihydroxy-6-heptenoate]) is a recently developed HMG-CoA reductase inhibitor that significantly reduces serum total cholesterol, LDL cholesterol, and triglycerides while modestly raising HDL cholesterol.28,29 We evaluated the effects of pitavastatin on the expression of CD36 by murine macrophages and demonstrate a novel mechanism by which pitavastatin inhibits CD36 expression, through PPARγ-dependent inhibition of CD36 gene transcription.

THP1 cells (a human monocytic cell line; ATCC, Rockville, Md) were cultured in RPMI 1640 complete medium. Cells were adjusted to a density of 300×103/cm2 in dishes before addition of PMA to drive the differentiation into macrophages. After the completion of differentiation (≈12 hours) and removal of PMA, cells were continued and treated in complete medium. These cells were used in fluorescence-activated cell sorter (FACS) studies because of the unavailability of antibodies to murine CD36.

Murine macrophages were obtained from C57BL/6 mice. Mice were injected intraperitoneally (3 mL per mouse) with 3% brewer thioglycollate medium (Difco). After 5 days, the mice were euthanized, and peritoneal macrophages were harvested by lavage. Cells were washed once with PBS and cultured in 60-mm dishes with complete RPMI medium for 4 hours. Medium containing floating cells was aspirated, and adherent cells were washed once with PBS, then cultured in complete medium.

Isolation of LDL and Preparation of OxLDL

LDL (1.019 to 1.063 g/mL) was isolated from normal human plasma by sequential ultracentrifugation, dialyzed against PBS containing 0.3 mmol/L EDTA, sterilized by filtration through a 0.22-μm filter, and stored under N2 gas at 4°C. Protein content was determined by the methods of Lowry.30 LDL was iodinated by the method of Bilheimer et al as described by Goldstein et al31 using carrier-free [125I]Na (Amersham Corp).

OxLDL was prepared as described previously.20 The purity and charge of both LDL and OxLDL were evaluated by examining electrophoretic migration in agarose gels. The degree of oxidation of LDL and OxLDL was determined by measuring the amount of thiobarbituric acid reactive substances (TBAR). LDL had TBAR values of <1 nmol/mg. OxLDL had TBAR values of >10 and <30 nmol/mg. All lipoproteins were used for experiments within 3 weeks after preparation.

Isolation of Total RNA, Purification of Poly(A+) RNA, and Northern Blotting

Cells were lysed in RNAzol B (Tel-Test, Inc), chloroform was extracted, and total cellular RNA was precipitated in isopropanol. After washing with 80% and 100% ethanol, the dried pellet of total RNA was dissolved in distilled water and quantified. The poly(A+) RNA was purified from ≈80 μg of total RNA with the PolyAT tract mRNA Isolation System III (Promega).

Poly(A+) RNA was loaded on 1% formaldehyde agarose gel. After electrophoresis, poly(A+) RNA was transferred to a Zeta-probe GT genomic-tested blotting membrane (Bio-Rad Laboratories) in 10×SSC by capillary force overnight. The blot was UV crosslinked for 2 minutes and prehybridized with Hybrisol I (Oncor, Inc) for 30 minutes before the addition of 32P randomly primed labeling probe for mouse CD36 or PPARγ or GAPDH. After overnight hybridization, the membrane was washed for 2×20 minutes with 2×SSC and 0.2% SDS and for 2×20 minutes with 0.2×SSC and 0.2% SDS at 55°C. The blot was autoradiographed by exposure to radiographic film (X-Omat AR, Kodak). Autoradiograms were assessed by densitometric scanning with a UMAX UC630 flatbed scanner attached to a Macintosh IIci (Apple Computer) running NIH Image software. The probe for mouse CD36 is an NsiI-BglII digest (base pairs 193 to 805). The template DNA for PPARγ was generated by reverse transcription-polymerase chain reaction (RT-PCR) based on the published sequences. The sequences of 5′ and 3′ oligonucleotides used for PPARγ were TCGGCGTTGTCATGATCCTC (121–141) and GGTTCATAAAAGCACGCTGG (551–571), respectively.

Flow Cytometry

After treatment, cells were lifted by addition of trypsin. Cells were washed 3 times with PBS. Approximately 1×106 cells were suspended in 300 μL of PBS containing 5% mouse serum and incubated for 30 minutes at room temperature with shaking. Cells then were added to 10 μL of mouse anti-human CD36 antibody conjugated to fluorescein isothiocyanate isomer 1 (FITC; Chemicon International Inc). After incubation with the antibody for 1 hour at room temperature, cells were washed 3 times with PBS. After suspension in PBS, cells were subjected to flow cytometric analysis with a Coulter FACScan.

Nuclear proteins (500 μg) from each sample were incubated with an antibody to mouse PPARγ antibody. Immunoabsorbed proteins were separated by SDS-PAGE and transferred onto nylon-enhanced nitrocellulose membrane, then analyzed by Western blot for phospho-PPARγ (PPARγ-Pi) by incubation with anti-phosphoserine antibodies. The nuclear proteins were also used to analyze PPARγ protein expression by SDS-PAGE/Western blot.

Results

Pitavastatin Downregulates CD36 Expression

To determine the effects of pitavastatin on macrophage expression of CD36, J774 cells were treated with various concentrations of pitavastatin for 24 hours. Total RNA was extracted and used to isolate Poly(A+) RNA. Northern blotting was used to determine expression of CD36 mRNA. Pitavastatin decreased CD36 expression in a dose-dependent manner (Figure 1A). At a concentration of 10 μmol/L, pitavastatin inhibited expression of CD36 mRNA by more than half. To demonstrate that pitavastatin altered expression of CD36 in primary cells in a manner similar to its effect on macrophage cell lines, we evaluated the effect of pitavastatin on CD36 expression in murine peritoneum-derived macrophages. Pitavastatin had similar effects on expression of CD36 mRNA in murine peritoneal macrophages (Figure 1B). To demonstrate that the statin effect on CD36 expression was not limited to pitavastatin, J774 cells were treated with pravastatin and simvastatin. Both statins decreased expression of CD36 in a dose-dependent manner (Figure 1C).

Figure 1. Pitavastatin decreases CD36 mRNA expression. A, J774 murine macrophages were cultured in complete RPMI medium until confluence, switched to serum-free medium, and treated with pitavastatin at indicated concentrations for 24 hours. Total RNA (100 μg) was used to isolate poly(A+) RNA. Poly(A+) RNA was transferred to a Zeta-probe GT genomic-tested blotting membrane after electrophoresis. The blot was UV crosslinked and hybridized overnight with 32P randomly primed cDNA probe for murine CD36. The blot was rehybridized with 32P-labeled GAPDH. Data are representative of 3 separate experiments. B, Murine peritoneal macrophages were treated with NK-104 at indicated concentrations for 16 hours in serum-free medium. Total RNA was collected and analyzed for CD36 expression as above. C, J774 cells were treated with pravastatin and simvastatin at the indicated concentrations for 24 hours. Expression of CD36 was determined by Northern blot as described above. Ctrl indicates control.

We next determined the effect of pitavastatin on CD36 protein expression. PMA-differentiated THP-1 cells (a human monocyte cell line) and human peripheral blood monocyte-derived macrophages were incubated with or without pitavastatin for 24 hours. CD36 surface protein expression was assessed by flow cytometry. Consistent with the effect of pitavastatin on CD36 mRNA, CD36 surface protein was substantially decreased (Figures 2A and 2B). To assess the functional significance of reduced expression of CD36 surface protein expression, we examined the binding/uptake of 125I-labeled OxLDL. Pitavastatin reduced both baseline binding/uptake of OxLDL and binding/uptake of OxLDL induced by a PPARγ ligand, prostaglandin J2 (PGJ2; data not shown).

Pitavastatin Decreases CD36 Expression at the Transcriptional Level and by Inactivating PPARγ

To determine whether pitavastatin decreased CD36 mRNA expression by reducing CD36 transcription or by altering CD36 mRNA stability, cells were treated with or without pitavastatin (10 μmol/L) in the presence of the transcriptional inhibitor actinomycin D. The half-life of CD36 was similar in both the presence and absence of pitavastatin (data not shown). Because CD36 RNA stability was not altered by pitavastatin, it is likely that pitavastatin downregulates CD36 mRNA at the transcriptional level.

To further investigate the mechanism by which pitavastatin regulates CD36 expression, we evaluated the effect of pitavastatin on PPARγ expression. Pitavastatin decreased expression of PPARγ mRNA (Figure 5A) and protein (Figure 5B) in a dose-dependent manner. The phosphorylation status of PPARγ has been shown to affect its transcription activity. PPARγ is a positive regulator for its target genes, whereas its phosphorylated form (PPARγ-Pi) is a negative regulator. Thus, the ratio of PPARγ-Pi to PPARγ is important for determining PPARγ-mediated transcriptional function. We evaluated PPARγ phosphorylation status in response to pitavastatin and found that expression of the nonphosphorylated form of PPARγ was decreased. Whereas the amount of PPARγ-Pi remained unchanged, the ratio of PPARγ-Pi to PPARγ was increased. This results in decreased PPARγ-dependent transcription and decreased expression of CD36. To determine the mechanism of PPARγ phosphorylation, we evaluated the effect of pitavastatin on p44/42 MAP kinase activity, which has been shown to phosphorylate serine residues in PPARγ. Beginning 4 hours after treatment with pitavastatin (and lasting for 24 hours), we observed increased p44/42 MAP kinase activity, as indicated by p44/42 MAP kinase phosphorylation (Figure 5C).

Figure 5. Pitavastatin downregulates PPARγ mRNA expression, increases PPARγ phosphorylation, and increases p44/42 MAP kinase activity. A, J774 macrophages were treated with pitavastatin at the indicated concentrations for 16 hours. Total RNA was extracted, and poly(A+) RNA was isolated. PPARγ mRNA was detected by Northern blot with a 32P randomly primed cDNA probe for murine PPARγ and GAPDH. Data are representative of 3 separate experiments. B, J774 macrophages were treated with pitavastatin at indicated concentrations for 16 hours. Nuclear proteins were extracted. After immunoprecipitation, PPARγ-Pi was detected by Western blot with anti-phosphoserine antibody. PPARγ was detected by anti-PPARγ antibody. Data are representative of 3 separate experiments. C, J774 macrophages were treated with pitavastatin (10 μmol/L) for indicated times. Cellular protein was extracted and analyzed for phosphorylated p44/42 MAP kinase (MAPK) by Western blot as described in Methods. Data are representative of 3 separate experiments.

Discussion

The present data demonstrate that pitavastatin inhibits expression of CD36 both by decreasing expression of PPARγ and through MAP kinase-mediated phosphorylation of PPARγ. This results in an increased ratio of phosphorylated PPARγ to nonphosphorylated PPARγ, which leads to decreased CD36 gene transcription.

PPARs become transcriptionally active when bound to ligand.24 Growth factors, such as epidermal growth factor and platelet-derived growth factor, have been shown to phosphorylate PPARγ via the MAP kinase signaling pathway and to decrease PPARγ transcriptional activity.27 The NH2-terminal domain of PPARγ contains a consensus MAP kinase site in a region conserved between PPARγ1 and PPARγ2 isoforms.33 PPARγ proteins migrate on immunoblots as closely spaced doublets, a pattern suggestive of phosphorylation.34,35 A putative MAP kinase site is phosphorylated by ERK2 and JNK.33 Phosphorylation significantly inhibits both ligand-independent and -dependent transcriptional activation by PPARγ.33 This repression is mediated by MAP kinase phosphorylation of Ser82 on PPARγ1.27 Mutation of the phosphorylated residue (Ser82) prevents PPARγ1 phosphorylation and the growth factor-mediated repression of PPARγ-dependent transcription. This phosphorylation-mediated transcriptional repression results from an alteration in the ability of PPARγ to become transcriptionally activated by ligand and is not due to a reduced capacity of the PPARγ · RXR complex to heterodimerize or recognize its DNA binding site.27 We have previously shown that both transforming growth factor-β36 and HDL32 induce MAP kinase-mediated phosphorylation of PPARγ.

The effect of statins on scavenger receptor expression has been evaluated previously. Lovastatin inhibited expression of both the type A scavenger receptor and CD36. Lovastatin decreased type A scavenger receptor mRNA in PMA-treated THP-1 cells.8 This inhibition was reversed by the addition of exogenous mevalonate.8 Similarly, lovastatin inhibited CD36 expression in human monocytic U937 cells, as measured by quantitative RT-PCR and FACS.9 Mevalonate completely reversed the effects of lovastatin, whereas excess LDL was only partially effective. Lovastatin was also shown to decrease expression of CD36 and the type A scavenger receptor in human blood-derived monocytes in culture for 2 and 5 days but not in more mature macrophages.37 Finally, monocyte-derived macrophages had decreased expression of CD36 and the type A scavenger receptor in hypercholesterolemic patients treated with atorvastatin relative to monocyte-derived macrophages from untreated patients.38 However, in these studies, no molecular mechanisms were identified by which statins decreased scavenger receptor expression.

In conclusion, the present study demonstrates that pitavastatin modulates phosphorylation and activity of PPARγ, which leads to decreased expression of CD36, a major macrophage scavenger receptor for oxidized lipids. The effect of pitavastatin on inhibition of CD36 expression may have relevance to both atherosclerotic foam cell formation mediated by CD36 and PPARγ and expression of other PPARγ-responsive inflammatory mediators expressed within vascular lesions.

Acknowledgments

This study was supported by a sponsored research agreement with Kowa Co, Ltd, Tokyo, Japan (Drs Hajjar and Gotto). Drs Hajjar and Gotto are consultants with Kowa Co, Ltd. Additional support was provided by the Abercrombie Foundation (Dr Gotto) and the Rosanne H. Silberman Foundation (Dr Nicholson).